707
Journal of Physiology (1990), 428, pp. 707-722 With 8 figures Printed in Great Britain
CHARACTERISTICS OF MINIATURE INHIBITORY POSTSYNAPTIC CURRENTS IN CAl PYRAMIDAL NEURONES OF RAT HIPPOCAMPUS
BY NICOLE ROPERT, RICHARD MILES AND HENRI KORN From the Laboratoire de Neurobiologie Cellulaire, Institut Pasteur, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France
(Received 31 January 1990) SUMMARY
1. Recordings were made in vitro from chloride-loaded CAI rat hippocampal pyramidal neurones in the presence of tetrodotoxin (TTX) to examine miniature inhibitory postsynaptic currents (IPSCs). 2. Most spontaneous synaptic events recorded before TTX was applied, and all events that were resolved in the presence of TTX, were blocked by the GABAA receptor antagonist bicuculline. 3. At 25 °C, averaged miniature IPSCs had a time to peak of about 3 ms and in most cases decayed with a single time constant close to 25 ms. 4. With a driving force for chloride ions between 70 and 80 mV, the mean miniature IPSC amplitude was 19-6-27-9 pA, yielding a conductance of 258-326 pS. The mean amplitude of unitary IPSCs recorded before TTX was applied was in the range of 31-73 pA. 5. When intervals between miniature IPSCs were compared with an exponential distribution, there was an excess of events at intervals shorter than 5 ms. Some individual events appeared to represent the nearly simultaneous release of two inhibitory quanta. 6. Miniature IPSC amplitude distributions were better fitted with the sum of two Gaussians than with one Gaussian. The variance in amplitude of a single quantal event exceeded that of the baseline noise. 7. Comparison of the conductance changes corresponding to the first Gaussian distribution with single GABA channel data suggests that one inhibitory quantum opens twelve to twenty chloride channels and that GABA molecules bind once to a postsynaptic receptor. INTRODUCTION
The observation that miniature endplate potentials occur in the absence of presynaptic activity suggested that chemical transmission was quantal at the neuromuscular junction (Fatt & Katz, 1952; Katz, 1969). These miniature events were thought to reflect the postsynaptic action of transmitter contained in a single vesicle and released by spontaneous exocytosis (Katz, 1978; Ceccarelli & Hurlbut, 1980; Van der Kloot, 1988). The persistence of miniature synaptic events while MS 8235
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708
708
ROPERT. R. MIILES AND H. KORN
action potentials were blocked in the presence of tetrodotoxin (TTX) has been taken as evidence that transmission is quantal at a number of central nervous system synapses (Brown, Wong & Prince, 1979: Takahashi. 1984; Kojima & Takahashi, 1985; Korn, Burnod & Faber, 1987; Bekkers & Stevens, 1989). In addition. the study of single quantal events can give direct access to the nature of interactions between postsynaptic receptors and transmitter released at a single site (Katz & Miledi, 1970; Anderson & Stevens, 1973). While miniature synaptic events corresponding to the release of acetylcholine (Fatt & Katz, 1952), glycine (Korn et al. 1987) and excitatory amino acids (Brown et al. 1979) have been resolved, the situation is less clear for the inhibitory neurotransmitter y-aminobutyric acid (GABA). Indeed it has been suggested that at some synapses GABA may be released by a carrier mechanism rather than in a quantal fashion (Schwartz, 1987). However, in hippocampal pyramidal cells fast GABA-dependent IPSPs with variable amplitude occur spontaneously (Alger & Nicoll, 1980; Collingridge, Gage & Robertson, 1984) and IPSPs evoked at single inhibitory connections appear to fluctuate in amplitude (Miles & Wong. 1984). Nevertheless it remains important to resolve single quantal events at a GABAmediated inhibitory synapse. In the present experiments, we attempted to record miniature IPSCs from chloride-filled CAI pyramidal cells in TTX. A comparison of the conductance of a single inhibitory quantum with that of single GABA channels suggests that transmitter released at a single site opens very few postsynaptic channels. Some of these results appeared previously in an abstract form (Ropert & Korn, 1988). METHODS
Transverse rat hippocampal slices (400-450 ,tm) were obtained from Sprague-Dawley male rats (50-200 g) decapitated with a guillotine. The brain was quickly removed, and the hippocampi were dissected in cold, oxygenated artificial cerebrospinal fluid (ACSF) and sliced using a Mclllwain tissue chopper. Slices were kept at room temperature (22-25 °C) in oxygenated (5 % C02-95 % 02) ACSF. The standard ACSF contained (in mm): NaCl, 125; KCl, 3 KH2PO4. 125; MgSO4, 2; CaCl2. 2; NaHCO3, 26; glucose, 11. All the recordings were performed from 1 to 9 h following the slice preparation. The recording chamber was a small (1 ml) chamber in which the slices were submerged and fixed to a nylon mesh by platinum wires. The ACSF was perfused bv gravity at a constant flow rate (2-2-5 ml/min). When the solution was switched to one containing tetrodotoxin (TTX), a blockade of spontaneous and evoked synaptic potentials and of fast sodium-dependent action potentials began within one minute and complete suppression was obtained in about two minutes. The following drugs were applied in the perfusion: tetrodotoxin (TTX. 0-4-08 ,UM); caesium chloride (2 mM); bicuculline methiodide (10 /M). All drugs were from Sigma. For fibre track stimulation, short voltage pulses (002-0{5 ms duration) were applied through bipolar stimulating electrodes made of two coated nickel-chrome wires (80 ,im in diameter) twisted together and cut at the tip. Stimulating electrodes were placed on the subiculum border of the alveus in the CA1 region in order to elicit recurrent IPSCs (Andersen, Eccles & Loyning, 1964). Intracellular recordings were obtained with 40-80 MQ pipettes filled with potassium chloride (2-8 M). Synaptic currents were measured using the discontinuous single-electrode voltage-clamp (dSEVC) method with an Axoclamp-2 preamplifier (Axon Instruments). The monitor signal was used to adjust the switching frequency (3-5 kHz) and the negative capacitance. The level of fluid covering the slices was reduced to minimize the time constant of the microelectrode. The output signal was filtered at 1 kHz. Inhibitory postsynaptic currents (IPSC) could be recorded with good
HIPPOCAMPAL MINL4TURE IPSC
709
control of the membrane potential (Johnston & Brown, 1984) in agreement with the observation that inhibitory svnaptic terminals surround the soma of CAI pvramidal cells (Ribak, Vaughn & Saito, 1978; Schwartzkroin & Mathers, 1978; Kawaguchi & Hama, 1988). Neurones were maintained at potentials between -80 and -90 mV in order to prevent firing of action potentials due to reversed IPSCs in chloride-loaded cells. Extracellular caesium (2 mM) was present in most recordings (Table 1) to block the inwardly rectifying potassium current, I., which reduces neuronal space constant in this potential range (Halliwell & Adams. 1982). In these conditions, the input conductance of the neurones was 30 % smaller than its normal value (Table 1) and constant over the whole stepping potential range (-80 to - 200 mV) used in these experiments. After penetrating a neurone. chloride loading was complete within 10-20 min as judged by an absence of further change in the reversal potential of the alveus-evoked IPSC or the amplitude of spontaneous and evoked IPSCs. Spontaneous unitary IPSCs were then recorded for 10-15 min. Tetrodotoxin (0-4-0-8 /M) was then added while monitoring sodium spikes elicited by current injection and synaptic activity evoked by alveus stimulation. Measurements of miniature IPSCs were made at least 5 min after spikes and alveus-evoked events were blocked. The current and voltage signals were amplified and displayed on an oscilloscope and a chart recorder (Gould 2400 S) and simultaneouslv recorded on a videotape. Evoked events were analysed on an IBM AT3 computer using a Tekmar acquisition board and the P-clamp software. A MINC8INDEC system was used for the measurements of spontaneous events. Spontaneous unitary IPSCs often appeared as bursts with IPSCs superimposed on the decay of a preceding event. The computer allowed the peak amplitude of the second event to be measured, taking into account the decay time constant and the time of oecurrence of the first IPSC (Korn et al. 1987). The background noise was estimated from traces without PSCs by measuring the difference in current between two cursors maintained at an interval equal to the time to peak of the IPSCs. The polarity of the IPSCs displayed from the MINC8-INDEC system (Figs 4. 5 and 7) is reversed: inward current is upward. RESULTS
Intracellular recordings were obtained from CAI pyramidal neurones with a stable resting membrane potential larger than -60 mV, an input resistance (Rm) larger than 30 MQ and an action potential amplitude larger than 80 mV. As CAI neurones were loaded with chloride, continuous depolarizing potentials became apparent. Under voltage clamp, spontaneous inward currents of variable amplitude with a fast rising phase and a slower decay were seen. These currents occurred as single events or more frequently in bursts. The frequency of spontaneous currents varied, ranging from 3-9 to 34 Hz, presumably according to the activity and number of inhibitory cells which made synapses with the recorded cell. Twenty-seven neurones with spontaneous synaptic activity were studied using the current-clamp method in twelve neurones and the dSEVC method for the other fifteen neurones. Measurements of individual synaptic events were made only from the dSEVC recordings.
Inhibitory nature of synaptic noise As both excitatory and inhibitory PSCs could participate in synaptic noise, we examined the effect of bicuculline (10 /JM, n = 9) on spontaneously occurring synaptic events. The cell shown in Fig. 1 exhibited typical synaptic noise with bursts of inward currents. With bicuculline, the frequency and amplitude of the spontaneous PSCs was reduced. Small inward currents could still be seen (Fig. iB); they were probably EPSCs. Their amplitude was smaller than 20 pA while the amplitude of bicuculline-sensitive PSCs ranged up to 390 pA. The frequency of spontaneous EPSCs was always lower than that of the IPSCs (Fig. 1). We did not attempt detailed
710
71'. ROPERT, R. MILES AND H. KORN A
Control
1~~~~~~~~~
V
10 jM-bicuculline
200 pA 20 mV
B 10 s
Fig. 1. Synaptic noise is predominantly inhibitory. In control conditions (A), spontaneous synaptic activity was seen as fast inward (downward) currents ranging predominantly from 10 to 30 pA. In the presence of bicuculline (10 /M), most synaptic noise was eliminated (B). Some inward currents (less than 10 pA) occurred at low frequency. Both the current (I) and the voltage (V) signals are shown. KCl (2-8 M) in recording pipette. Holding potential, -88 mV. Extracellular caesium, 2 mm. A
Control
B 0.5,uM-TTX _
C 0-5 ,uM-TTX and 10 pM-bicuculline
10 s Fig. 2. Spontaneous inhibitory postsynaptic currents (IPSCs). In control conditions (A), continuous inward (downward) current fluctuations were observed. In B, 20 min after TTX application (0-5 /M), some small (10-90 pA) inward currents remained. In the presence of TTX, bicuculline (10 ,UM) suppressed inward currents (C). KCl (2-8 M) in recording pipette. Extracellular caesium, 2 mm. Holding potential, -78 mV.
e-~ ~L°0_
HIPPOCAMPAL MINIATURE IPSC 711 measurements of EPSCs. It is concluded that in CAI pyramidal neurones the synaptic noise is mainly inhibitory. Occurrence of miniature IPSCs Tetrodotoxin (0-40-8 jtM) was applied to twenty-one neurones to block sodiumdependent action potentials so that miniature synaptic events could be examined. In A
C
0M
Control
>. 006:
-~~~~~0-4 I
~~~0
4
B
100 ms
D 0 16
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-50
0
50
100 150 200
250
Amplitude (pA) 0 5 pM-TTX
0.12
o0.08 0~
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0 -50
0
50
100
150 200
250
Amplitude (PA)
Fig. 3. Amplitude distributions of miniature and unitary spontaneous IPSCs. The activity of the same neurone as in Fig. 2 is presented at a faster speed in control conditions (A) and in the presence of 0-5 /Sm-TTX (B). Calibration, 200 pA and 100 ms in A and B. Spontaneous currents had a fast rising phase and a slower decay. During control (A), their amplitude fluctuated more than inITTX. Amplitude histograms were built as described in the Methods section, during the control (C) and in the presence of TTX (D). With TITX (D), events were smaller and more homogeneous. In control 2015 events occurring during 1 min were measured. In TX, 585 events were measured from 6 mi. sixteen neurones, inward currents were still present after the alveus-evoked synaptic response and the direct action potentials had been blocked for 5-30 min. In the neurone shown in Fig. 2, the mean frequency of inward currents dropped from 34-2 to 1-6 Hz with TITX (0-5 gim). During the control period, currents tended to appear in bursts at regular intervals (every 2 s); during the burst, the frequency could reach 200 Hz and individual events summated. Mfter TTX, events were more regularly distributed and their amplitude was more constant. These TTX-resistant events could correspond to the spontaneous release of GABA or glutamate. However, since the GABAA antagonist bicuculline (10 gm) eliminated all currents in a reversible manner (n = 3/3; Fig. 2), we conclude that the TTXresistant inward currents represent miniature IPSCs. Miniature EPSOs could not be resolved in the presence of bicuculline and TTX.
712
N ROPERT, R. MILES AND H. KORN
Figure 3 compares unitary and miniature IPSCs recorded from the same cell. The mean amplitude of spontaneous IPSCs was reduced from 58 1 + 42-6 pA (mean + S.D.; range, 9-346 pA, n = 2015) in control (Fig. 3A and C) to 27-9 + 12 1 pA (range 10-189 pA, n = 585) in TTX (Fig. 3B and D). However, the peaks of both amplitude A
50 ms
-1
B
1
100
10) 0. .
0.
*
0-
E ~0
4 6 Time (min) Fig. 4. Stationarity of miniature IPSCs. A shows typical miniature IPSCs from a single neurone. In this and the following figures, inward currents are upward. Holding potential, -96 mV. Extracellular caesium, 2 mm. In B, the amplitude of all miniature IPSCs recorded over a 6 min period is plotted versus time. The straight line is the regression curve of the IPSC amplitude versus time with a slope equal to zero.
0
2
distributions (Fig. 3A and B) were at a similar amplitude close to 25 pA. The unitary and the TTX-resistant inward currents have a similar time course. Their time to peak in control conditions (3-1 + 1 1 ms, n = 2015) was slightly longer than its value in TTX (2-8 + 0-8 ms, n = 585) since large unitary IPSCs tended to have a longer time to peak. The time constant of decay of both the unitary and the miniature IPSCs was similar (range 15-28 ms).
Properties of miniature IPSCs These data suggest that all inward currents recorded from CAI neurones, in the presence of TTX, correspond to the action potential-independent release of GABA from terminals of inhibitory cells. We investigated their time course and their amplitude distribution.
HIPPOCAMPAL MIiNIATURE IPSC
713
B 100
80 c
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8
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20 40 60 80 Amplitude (pA) Fig. 5. Shape of the miniature IPSCs. The trace in A is an average of 464 miniature IPSCs (inward current is upward). Its amplitude was 25-4 pA and its time to peak was 3-25 ms. Its decay followed a single exponential with a time constant of 28 ms. B, the time to peak distribution for miniature IPSCs, was fitted by a Gaussian function (mean = 2-37 ms, S.D. = 0-57 ms). C shows a plot of the miniature IPSC time to peak versus their amplitude. A small positive correlation was seen. The time to peak increased by 0-42 ms every 10 pA with a correlation coefficient of 0-62. 0
The miniature IPSCs had a steep rising phase and a slower decay (Fig. 4A). We could detect with certainty events larger than twice the standard deviation of the background noise which was measured from traces without miniature IPSCs. Although sodium-dependent action potential and evoked synaptic events were blocked, we wished to ensure that there was no systematic change in the miniature IPSCs. The amplitude of individual events was plotted as a function of time (Fig. 4B). Although fluctuations occurred, the mean stayed constant as shown by a slope of zero for the regression of amplitude against time. Further analysis was carried out on four cells where miniature IPSCs were stationary for 15-30 min. Time course of TTX-resistant inhibitory events The time course and amplitude of miniature synaptic events reflect the number and duration of openings of postsynaptic channels following single exocytosis (Katz
714
N. ROPERT, R. MILES AND H. KORN
& Miledi, 1970). For each neurone, the miniature IPSCs were averaged to obtain a mean TTX-resistant IPSC (Fig. 5A) with time to peak ranging from 2-75 to 4 ms. In three neurones, their decay could be fitted with a single exponential with a time constant of 21-28 ms (Fig. 5A); in the fourth neurone, the averaged IPSC decayed with a fast early component (6 ms) and a slower late decay of 15 ms (Table 1). Figure 5B TABLE 1. Characteristics of miniature IPSCs Conductance Miniature IPSC (nS) Cell g ev i(n) Decay VH no. Cs' (mV) G1 (mV) (pA) (pS) (Ms) G2 1 N -85 19 62 (517) 6-15 2 N -97 -12 13 N 3 -76 15 -37 Y 4 12 24 -96 8 -20 19-6 (349) 258 Y 5 12 -83 9 -5 Y 6 -78 9 0 25-4 (464) 326 28 7 6 Y -86 21P8 (369) 21 Y 8 -101 7 -33 Y 10 2 -88 7 -53 11 Y -85 8 4 20 Neurones were held at negative potentials (VH), in the presence (Y) or the absence (N) of caesium (2 mM). The input conductance was measured in the absence (G1) and in the presence (G2) of caesium. The IPSC reversal potential (VKV) was measured. The averaged miniature IPSC amplitude (i) was measured in four neurones. n was the number of events measured. The miniature conductance (g) was calculated using the equation g = i/(VH- V,ev). The time constant of decay for the miniature IPSC was measured from averaged miniature IPSCs and averaged unitary IPSCs (cell no. 11).
shows there were fluctuations in the time to peak of miniature IPSCs. In this cell the distribution was fitted with a Gaussian curve with a coefficient of variation 0-25. When time to peak for individual events was plotted against their amplitude, we observed in all cells a weak positive correlation (Fig. 5C). This was unexpected since if electrotonic factors accounted for fluctuations in miniature amplitude smaller IPSCs should have a longer time to peak, assuming the shape of events does not vary with location of synapses. In four neurones held between -96 and -78 mV, the averaged amplitude of miniature IPSCs ranged from 19-6 to 62 pA. From this amplitude and the IPSC driving force, the conductance of an averaged miniature IPSC (g) could be estimated. It was difficult to determine the reversal potential of the miniature events because they were small and appeared at a low frequency. Therefore we measured the IPSC reversal potential before applying TTX. Low-intensity alvear stimulation was used to evoke a recurrent IPSC (Dingledine & Langmoen, 1980) which was completely blocked by bicuculline (10 /M, n = 4). The amplitude of these IPSCs ranged from 1 12 to 0-17 nA (0-42 + 0 1 nA, mean + S.D., n = 9). The reversal of the recurrent IPSC was measured by stepping the potential to various levels from a holding potential close to -90 mV to avoid variations in chloride loading. The mean estimated reversal potential was - 19-5 + 7 mV in eight neurones (Table 1). Using this reversal
HIPPOCAMPAL MINIATURE IPSC 715 potential, the conductance of the averaged TTX-resistant events was calculated in two neurones from the averaged quantal current to obtain values of 258 and 326 pS. Frequency and interval distributions of the miniature IPSCs Miniature IPSCs were not observed in five neurones. In the other sixteen cells, their frequency varied between 1-2 and 1'8 Hz and was constant for a single neurone A 4
2-
.
0*
vA.B.6 0
2
B
150: >
100_
cr
50-
0,
C 240
4
6
Time (min)
0
2
0
2
6 Interval (s)
8
10
4
8
10
4
o 160
= 80 U-
6
Interval (s)
Fig. 6. Frequency and interval distributions of miniature IPSCs. A, starting 4 min after the beginning of TTX (0-6 ,M), the number of miniature IPSCs occurring every 3-75 s was measured for 6 min. This frequency was stable and averaged 1-24 Hz. B shows the distribution of intervals between IPSCs. In this neurone the interval distribution could be fitted with a single exponential. C shows another neurone where the experimental data deviated from a single exponential fit at short intervals.
(Fig. 6A). An exponential distribution of intervals between miniature events has been taken as evidence for independent release (Fatt & Katz, 1952). In one neurone, the interval distribution followed a single exponential (Fig. 6B). In the other three neurones, there was a small excess of events at short intervals (Fig. 6C) possibly indicating subtle deviation from a random process. We therefore examined miniature events which were separated by short intervals. Figure 7A shows, from the same cell, a single event (A1), two events separated by short intervals (A2 and A3) and an apparent double event (A4). Similar observations were made in all cells. These coincidences may have occurred by chance. To investigate this point, we examined the distribution of short intervals (less than
716
N. ROPERT, R. MILES AND H. KORN
50 ms) between IPSCs. Figure 7D shows that many more events fell in the zero to 5 ms intervals than in any other 5 ms interval. It appears that the probability of an IPSC increases for a very short time following a preceding miniature event although it is unclear whether both events represent release from the same site.
B 2t_
40>
3_
Journal of Physiology (1990), 428, pp. 707-722 With 8 figures Printed in Great Britain
CHARACTERISTICS OF MINIATURE INHIBITORY POSTSYNAPTIC CURRENTS IN CAl PYRAMIDAL NEURONES OF RAT HIPPOCAMPUS
BY NICOLE ROPERT, RICHARD MILES AND HENRI KORN From the Laboratoire de Neurobiologie Cellulaire, Institut Pasteur, 25 Rue du Docteur Roux, 75724 Paris Cedex 15, France
(Received 31 January 1990) SUMMARY
1. Recordings were made in vitro from chloride-loaded CAI rat hippocampal pyramidal neurones in the presence of tetrodotoxin (TTX) to examine miniature inhibitory postsynaptic currents (IPSCs). 2. Most spontaneous synaptic events recorded before TTX was applied, and all events that were resolved in the presence of TTX, were blocked by the GABAA receptor antagonist bicuculline. 3. At 25 °C, averaged miniature IPSCs had a time to peak of about 3 ms and in most cases decayed with a single time constant close to 25 ms. 4. With a driving force for chloride ions between 70 and 80 mV, the mean miniature IPSC amplitude was 19-6-27-9 pA, yielding a conductance of 258-326 pS. The mean amplitude of unitary IPSCs recorded before TTX was applied was in the range of 31-73 pA. 5. When intervals between miniature IPSCs were compared with an exponential distribution, there was an excess of events at intervals shorter than 5 ms. Some individual events appeared to represent the nearly simultaneous release of two inhibitory quanta. 6. Miniature IPSC amplitude distributions were better fitted with the sum of two Gaussians than with one Gaussian. The variance in amplitude of a single quantal event exceeded that of the baseline noise. 7. Comparison of the conductance changes corresponding to the first Gaussian distribution with single GABA channel data suggests that one inhibitory quantum opens twelve to twenty chloride channels and that GABA molecules bind once to a postsynaptic receptor. INTRODUCTION
The observation that miniature endplate potentials occur in the absence of presynaptic activity suggested that chemical transmission was quantal at the neuromuscular junction (Fatt & Katz, 1952; Katz, 1969). These miniature events were thought to reflect the postsynaptic action of transmitter contained in a single vesicle and released by spontaneous exocytosis (Katz, 1978; Ceccarelli & Hurlbut, 1980; Van der Kloot, 1988). The persistence of miniature synaptic events while MS 8235
23-2
708
708
ROPERT. R. MIILES AND H. KORN
action potentials were blocked in the presence of tetrodotoxin (TTX) has been taken as evidence that transmission is quantal at a number of central nervous system synapses (Brown, Wong & Prince, 1979: Takahashi. 1984; Kojima & Takahashi, 1985; Korn, Burnod & Faber, 1987; Bekkers & Stevens, 1989). In addition. the study of single quantal events can give direct access to the nature of interactions between postsynaptic receptors and transmitter released at a single site (Katz & Miledi, 1970; Anderson & Stevens, 1973). While miniature synaptic events corresponding to the release of acetylcholine (Fatt & Katz, 1952), glycine (Korn et al. 1987) and excitatory amino acids (Brown et al. 1979) have been resolved, the situation is less clear for the inhibitory neurotransmitter y-aminobutyric acid (GABA). Indeed it has been suggested that at some synapses GABA may be released by a carrier mechanism rather than in a quantal fashion (Schwartz, 1987). However, in hippocampal pyramidal cells fast GABA-dependent IPSPs with variable amplitude occur spontaneously (Alger & Nicoll, 1980; Collingridge, Gage & Robertson, 1984) and IPSPs evoked at single inhibitory connections appear to fluctuate in amplitude (Miles & Wong. 1984). Nevertheless it remains important to resolve single quantal events at a GABAmediated inhibitory synapse. In the present experiments, we attempted to record miniature IPSCs from chloride-filled CAI pyramidal cells in TTX. A comparison of the conductance of a single inhibitory quantum with that of single GABA channels suggests that transmitter released at a single site opens very few postsynaptic channels. Some of these results appeared previously in an abstract form (Ropert & Korn, 1988). METHODS
Transverse rat hippocampal slices (400-450 ,tm) were obtained from Sprague-Dawley male rats (50-200 g) decapitated with a guillotine. The brain was quickly removed, and the hippocampi were dissected in cold, oxygenated artificial cerebrospinal fluid (ACSF) and sliced using a Mclllwain tissue chopper. Slices were kept at room temperature (22-25 °C) in oxygenated (5 % C02-95 % 02) ACSF. The standard ACSF contained (in mm): NaCl, 125; KCl, 3 KH2PO4. 125; MgSO4, 2; CaCl2. 2; NaHCO3, 26; glucose, 11. All the recordings were performed from 1 to 9 h following the slice preparation. The recording chamber was a small (1 ml) chamber in which the slices were submerged and fixed to a nylon mesh by platinum wires. The ACSF was perfused bv gravity at a constant flow rate (2-2-5 ml/min). When the solution was switched to one containing tetrodotoxin (TTX), a blockade of spontaneous and evoked synaptic potentials and of fast sodium-dependent action potentials began within one minute and complete suppression was obtained in about two minutes. The following drugs were applied in the perfusion: tetrodotoxin (TTX. 0-4-08 ,UM); caesium chloride (2 mM); bicuculline methiodide (10 /M). All drugs were from Sigma. For fibre track stimulation, short voltage pulses (002-0{5 ms duration) were applied through bipolar stimulating electrodes made of two coated nickel-chrome wires (80 ,im in diameter) twisted together and cut at the tip. Stimulating electrodes were placed on the subiculum border of the alveus in the CA1 region in order to elicit recurrent IPSCs (Andersen, Eccles & Loyning, 1964). Intracellular recordings were obtained with 40-80 MQ pipettes filled with potassium chloride (2-8 M). Synaptic currents were measured using the discontinuous single-electrode voltage-clamp (dSEVC) method with an Axoclamp-2 preamplifier (Axon Instruments). The monitor signal was used to adjust the switching frequency (3-5 kHz) and the negative capacitance. The level of fluid covering the slices was reduced to minimize the time constant of the microelectrode. The output signal was filtered at 1 kHz. Inhibitory postsynaptic currents (IPSC) could be recorded with good
HIPPOCAMPAL MINL4TURE IPSC
709
control of the membrane potential (Johnston & Brown, 1984) in agreement with the observation that inhibitory svnaptic terminals surround the soma of CAI pvramidal cells (Ribak, Vaughn & Saito, 1978; Schwartzkroin & Mathers, 1978; Kawaguchi & Hama, 1988). Neurones were maintained at potentials between -80 and -90 mV in order to prevent firing of action potentials due to reversed IPSCs in chloride-loaded cells. Extracellular caesium (2 mM) was present in most recordings (Table 1) to block the inwardly rectifying potassium current, I., which reduces neuronal space constant in this potential range (Halliwell & Adams. 1982). In these conditions, the input conductance of the neurones was 30 % smaller than its normal value (Table 1) and constant over the whole stepping potential range (-80 to - 200 mV) used in these experiments. After penetrating a neurone. chloride loading was complete within 10-20 min as judged by an absence of further change in the reversal potential of the alveus-evoked IPSC or the amplitude of spontaneous and evoked IPSCs. Spontaneous unitary IPSCs were then recorded for 10-15 min. Tetrodotoxin (0-4-0-8 /M) was then added while monitoring sodium spikes elicited by current injection and synaptic activity evoked by alveus stimulation. Measurements of miniature IPSCs were made at least 5 min after spikes and alveus-evoked events were blocked. The current and voltage signals were amplified and displayed on an oscilloscope and a chart recorder (Gould 2400 S) and simultaneouslv recorded on a videotape. Evoked events were analysed on an IBM AT3 computer using a Tekmar acquisition board and the P-clamp software. A MINC8INDEC system was used for the measurements of spontaneous events. Spontaneous unitary IPSCs often appeared as bursts with IPSCs superimposed on the decay of a preceding event. The computer allowed the peak amplitude of the second event to be measured, taking into account the decay time constant and the time of oecurrence of the first IPSC (Korn et al. 1987). The background noise was estimated from traces without PSCs by measuring the difference in current between two cursors maintained at an interval equal to the time to peak of the IPSCs. The polarity of the IPSCs displayed from the MINC8-INDEC system (Figs 4. 5 and 7) is reversed: inward current is upward. RESULTS
Intracellular recordings were obtained from CAI pyramidal neurones with a stable resting membrane potential larger than -60 mV, an input resistance (Rm) larger than 30 MQ and an action potential amplitude larger than 80 mV. As CAI neurones were loaded with chloride, continuous depolarizing potentials became apparent. Under voltage clamp, spontaneous inward currents of variable amplitude with a fast rising phase and a slower decay were seen. These currents occurred as single events or more frequently in bursts. The frequency of spontaneous currents varied, ranging from 3-9 to 34 Hz, presumably according to the activity and number of inhibitory cells which made synapses with the recorded cell. Twenty-seven neurones with spontaneous synaptic activity were studied using the current-clamp method in twelve neurones and the dSEVC method for the other fifteen neurones. Measurements of individual synaptic events were made only from the dSEVC recordings.
Inhibitory nature of synaptic noise As both excitatory and inhibitory PSCs could participate in synaptic noise, we examined the effect of bicuculline (10 /JM, n = 9) on spontaneously occurring synaptic events. The cell shown in Fig. 1 exhibited typical synaptic noise with bursts of inward currents. With bicuculline, the frequency and amplitude of the spontaneous PSCs was reduced. Small inward currents could still be seen (Fig. iB); they were probably EPSCs. Their amplitude was smaller than 20 pA while the amplitude of bicuculline-sensitive PSCs ranged up to 390 pA. The frequency of spontaneous EPSCs was always lower than that of the IPSCs (Fig. 1). We did not attempt detailed
710
71'. ROPERT, R. MILES AND H. KORN A
Control
1~~~~~~~~~
V
10 jM-bicuculline
200 pA 20 mV
B 10 s
Fig. 1. Synaptic noise is predominantly inhibitory. In control conditions (A), spontaneous synaptic activity was seen as fast inward (downward) currents ranging predominantly from 10 to 30 pA. In the presence of bicuculline (10 /M), most synaptic noise was eliminated (B). Some inward currents (less than 10 pA) occurred at low frequency. Both the current (I) and the voltage (V) signals are shown. KCl (2-8 M) in recording pipette. Holding potential, -88 mV. Extracellular caesium, 2 mm. A
Control
B 0.5,uM-TTX _
C 0-5 ,uM-TTX and 10 pM-bicuculline
10 s Fig. 2. Spontaneous inhibitory postsynaptic currents (IPSCs). In control conditions (A), continuous inward (downward) current fluctuations were observed. In B, 20 min after TTX application (0-5 /M), some small (10-90 pA) inward currents remained. In the presence of TTX, bicuculline (10 ,UM) suppressed inward currents (C). KCl (2-8 M) in recording pipette. Extracellular caesium, 2 mm. Holding potential, -78 mV.
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HIPPOCAMPAL MINIATURE IPSC 711 measurements of EPSCs. It is concluded that in CAI pyramidal neurones the synaptic noise is mainly inhibitory. Occurrence of miniature IPSCs Tetrodotoxin (0-40-8 jtM) was applied to twenty-one neurones to block sodiumdependent action potentials so that miniature synaptic events could be examined. In A
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Amplitude (pA) 0 5 pM-TTX
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100
150 200
250
Amplitude (PA)
Fig. 3. Amplitude distributions of miniature and unitary spontaneous IPSCs. The activity of the same neurone as in Fig. 2 is presented at a faster speed in control conditions (A) and in the presence of 0-5 /Sm-TTX (B). Calibration, 200 pA and 100 ms in A and B. Spontaneous currents had a fast rising phase and a slower decay. During control (A), their amplitude fluctuated more than inITTX. Amplitude histograms were built as described in the Methods section, during the control (C) and in the presence of TTX (D). With TITX (D), events were smaller and more homogeneous. In control 2015 events occurring during 1 min were measured. In TX, 585 events were measured from 6 mi. sixteen neurones, inward currents were still present after the alveus-evoked synaptic response and the direct action potentials had been blocked for 5-30 min. In the neurone shown in Fig. 2, the mean frequency of inward currents dropped from 34-2 to 1-6 Hz with TITX (0-5 gim). During the control period, currents tended to appear in bursts at regular intervals (every 2 s); during the burst, the frequency could reach 200 Hz and individual events summated. Mfter TTX, events were more regularly distributed and their amplitude was more constant. These TTX-resistant events could correspond to the spontaneous release of GABA or glutamate. However, since the GABAA antagonist bicuculline (10 gm) eliminated all currents in a reversible manner (n = 3/3; Fig. 2), we conclude that the TTXresistant inward currents represent miniature IPSCs. Miniature EPSOs could not be resolved in the presence of bicuculline and TTX.
712
N ROPERT, R. MILES AND H. KORN
Figure 3 compares unitary and miniature IPSCs recorded from the same cell. The mean amplitude of spontaneous IPSCs was reduced from 58 1 + 42-6 pA (mean + S.D.; range, 9-346 pA, n = 2015) in control (Fig. 3A and C) to 27-9 + 12 1 pA (range 10-189 pA, n = 585) in TTX (Fig. 3B and D). However, the peaks of both amplitude A
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4 6 Time (min) Fig. 4. Stationarity of miniature IPSCs. A shows typical miniature IPSCs from a single neurone. In this and the following figures, inward currents are upward. Holding potential, -96 mV. Extracellular caesium, 2 mm. In B, the amplitude of all miniature IPSCs recorded over a 6 min period is plotted versus time. The straight line is the regression curve of the IPSC amplitude versus time with a slope equal to zero.
0
2
distributions (Fig. 3A and B) were at a similar amplitude close to 25 pA. The unitary and the TTX-resistant inward currents have a similar time course. Their time to peak in control conditions (3-1 + 1 1 ms, n = 2015) was slightly longer than its value in TTX (2-8 + 0-8 ms, n = 585) since large unitary IPSCs tended to have a longer time to peak. The time constant of decay of both the unitary and the miniature IPSCs was similar (range 15-28 ms).
Properties of miniature IPSCs These data suggest that all inward currents recorded from CAI neurones, in the presence of TTX, correspond to the action potential-independent release of GABA from terminals of inhibitory cells. We investigated their time course and their amplitude distribution.
HIPPOCAMPAL MIiNIATURE IPSC
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20 40 60 80 Amplitude (pA) Fig. 5. Shape of the miniature IPSCs. The trace in A is an average of 464 miniature IPSCs (inward current is upward). Its amplitude was 25-4 pA and its time to peak was 3-25 ms. Its decay followed a single exponential with a time constant of 28 ms. B, the time to peak distribution for miniature IPSCs, was fitted by a Gaussian function (mean = 2-37 ms, S.D. = 0-57 ms). C shows a plot of the miniature IPSC time to peak versus their amplitude. A small positive correlation was seen. The time to peak increased by 0-42 ms every 10 pA with a correlation coefficient of 0-62. 0
The miniature IPSCs had a steep rising phase and a slower decay (Fig. 4A). We could detect with certainty events larger than twice the standard deviation of the background noise which was measured from traces without miniature IPSCs. Although sodium-dependent action potential and evoked synaptic events were blocked, we wished to ensure that there was no systematic change in the miniature IPSCs. The amplitude of individual events was plotted as a function of time (Fig. 4B). Although fluctuations occurred, the mean stayed constant as shown by a slope of zero for the regression of amplitude against time. Further analysis was carried out on four cells where miniature IPSCs were stationary for 15-30 min. Time course of TTX-resistant inhibitory events The time course and amplitude of miniature synaptic events reflect the number and duration of openings of postsynaptic channels following single exocytosis (Katz
714
N. ROPERT, R. MILES AND H. KORN
& Miledi, 1970). For each neurone, the miniature IPSCs were averaged to obtain a mean TTX-resistant IPSC (Fig. 5A) with time to peak ranging from 2-75 to 4 ms. In three neurones, their decay could be fitted with a single exponential with a time constant of 21-28 ms (Fig. 5A); in the fourth neurone, the averaged IPSC decayed with a fast early component (6 ms) and a slower late decay of 15 ms (Table 1). Figure 5B TABLE 1. Characteristics of miniature IPSCs Conductance Miniature IPSC (nS) Cell g ev i(n) Decay VH no. Cs' (mV) G1 (mV) (pA) (pS) (Ms) G2 1 N -85 19 62 (517) 6-15 2 N -97 -12 13 N 3 -76 15 -37 Y 4 12 24 -96 8 -20 19-6 (349) 258 Y 5 12 -83 9 -5 Y 6 -78 9 0 25-4 (464) 326 28 7 6 Y -86 21P8 (369) 21 Y 8 -101 7 -33 Y 10 2 -88 7 -53 11 Y -85 8 4 20 Neurones were held at negative potentials (VH), in the presence (Y) or the absence (N) of caesium (2 mM). The input conductance was measured in the absence (G1) and in the presence (G2) of caesium. The IPSC reversal potential (VKV) was measured. The averaged miniature IPSC amplitude (i) was measured in four neurones. n was the number of events measured. The miniature conductance (g) was calculated using the equation g = i/(VH- V,ev). The time constant of decay for the miniature IPSC was measured from averaged miniature IPSCs and averaged unitary IPSCs (cell no. 11).
shows there were fluctuations in the time to peak of miniature IPSCs. In this cell the distribution was fitted with a Gaussian curve with a coefficient of variation 0-25. When time to peak for individual events was plotted against their amplitude, we observed in all cells a weak positive correlation (Fig. 5C). This was unexpected since if electrotonic factors accounted for fluctuations in miniature amplitude smaller IPSCs should have a longer time to peak, assuming the shape of events does not vary with location of synapses. In four neurones held between -96 and -78 mV, the averaged amplitude of miniature IPSCs ranged from 19-6 to 62 pA. From this amplitude and the IPSC driving force, the conductance of an averaged miniature IPSC (g) could be estimated. It was difficult to determine the reversal potential of the miniature events because they were small and appeared at a low frequency. Therefore we measured the IPSC reversal potential before applying TTX. Low-intensity alvear stimulation was used to evoke a recurrent IPSC (Dingledine & Langmoen, 1980) which was completely blocked by bicuculline (10 /M, n = 4). The amplitude of these IPSCs ranged from 1 12 to 0-17 nA (0-42 + 0 1 nA, mean + S.D., n = 9). The reversal of the recurrent IPSC was measured by stepping the potential to various levels from a holding potential close to -90 mV to avoid variations in chloride loading. The mean estimated reversal potential was - 19-5 + 7 mV in eight neurones (Table 1). Using this reversal
HIPPOCAMPAL MINIATURE IPSC 715 potential, the conductance of the averaged TTX-resistant events was calculated in two neurones from the averaged quantal current to obtain values of 258 and 326 pS. Frequency and interval distributions of the miniature IPSCs Miniature IPSCs were not observed in five neurones. In the other sixteen cells, their frequency varied between 1-2 and 1'8 Hz and was constant for a single neurone A 4
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Interval (s)
Fig. 6. Frequency and interval distributions of miniature IPSCs. A, starting 4 min after the beginning of TTX (0-6 ,M), the number of miniature IPSCs occurring every 3-75 s was measured for 6 min. This frequency was stable and averaged 1-24 Hz. B shows the distribution of intervals between IPSCs. In this neurone the interval distribution could be fitted with a single exponential. C shows another neurone where the experimental data deviated from a single exponential fit at short intervals.
(Fig. 6A). An exponential distribution of intervals between miniature events has been taken as evidence for independent release (Fatt & Katz, 1952). In one neurone, the interval distribution followed a single exponential (Fig. 6B). In the other three neurones, there was a small excess of events at short intervals (Fig. 6C) possibly indicating subtle deviation from a random process. We therefore examined miniature events which were separated by short intervals. Figure 7A shows, from the same cell, a single event (A1), two events separated by short intervals (A2 and A3) and an apparent double event (A4). Similar observations were made in all cells. These coincidences may have occurred by chance. To investigate this point, we examined the distribution of short intervals (less than
716
N. ROPERT, R. MILES AND H. KORN
50 ms) between IPSCs. Figure 7D shows that many more events fell in the zero to 5 ms intervals than in any other 5 ms interval. It appears that the probability of an IPSC increases for a very short time following a preceding miniature event although it is unclear whether both events represent release from the same site.
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